The redox state of a cell refers to the balance between oxidative processes and reducing processes. The energy released by oxidative processes is used by the cell to build cellular and tissue structures, and to operate and maintain such structures. The term redox state has typically been used to refer to two molecules between which electrons may be traded, and which are referred to as a “redox couple”. An example of such a couple is made up of the two molecules glutathione (GSH) and its oxidized form, glutathione disulfide (GSSG), which help to determine the balance between oxidative and reducing processes, and hence the redox state or environment of the cell. Another redox couple comprises NADPH and NADP+. The balance between the oxidized and reduced forms of these couples may have many important biological effects, particularly with regard to the growth and proliferation of the cell.
Without wishing to rule out other mechanisms, it can be assumed that the redox state of the cell has some measure of control over the proliferative behavior of the cell, and in particular to the induction of cessation of cell proliferation (CCP), as explained in greater detail below.
One way to describe the redox state or environment of the cell is through the Nernst Equation. Changes in the intracellular redox potential, E, are, according to the Nernst equation, proportional to changes in log {[GSH]2/[GSSG]}, where [GSH] and [GSSG] are the concentrations of GSH and GSSG, respectively. As [GSH] decreases, E increases (Hutter et al., 1997).
Decreasing the level of GSH increases the redox potential of the cell, and has been observed to lower the rate of cell proliferation. Normal actively proliferating (foreskin) fibroblasts have been observed to have an average E of about −222 mV, which is about 10 mV lower than that observed for neoplastic fibrosarcoma cells, where the average E has been observed to be about −211 mV (Hutter et al., 1997). Proliferative behavior appears to be associated with the redox potential of the cell. Decreasing the level of GSH increases the redox potential of a cell, and has been shown to result in a decrease, or cessation of, cell proliferation. Again, without limiting the process to a single mechanism, we suggest that such behavior is at least partially mediated through effects on the retinoblastoma (RB) protein, considered to be a master regulator of cell cycle, differentiation and apoptosis.
The human RB protein is a nuclear phosphoprotein spanning 928 amino acids in length that is expressed in every tissue type examined. This protein appears to be the major player in a regulatory circuit in the late G1 (growth) phase, the so-called restriction point R, that defines a timepoint in G1 at which cells are committed to enter S (DNA replication) phase and no longer respond to growth conditions. Moreover, RB is involved in regulating an elusive switch point between cell cycle, differentiation and apoptosis.
Functional interactions exist between RB and the three D cyclins, together with their associated kinases. Cyclins function to activate cyclin-dependent kinases, which facilitate adding phosphates onto other molecules that play a role in cell-cycle progression. The phosphorylation of RB, via cyclin-dependent kinases, correlates with an inactivation of its ability to arrest cellular division. Specifically, if RB is inactivated, a cell will proceed through the cell cycle, multiplying unchecked until the RB is again activated. Herein lie the implications for cancer biology.
When the GSH concentration in NK3.3 cells is sufficiently decreased, and hence E is sufficiently increased, the RB protein in these cells cannot be phosphorylated and the cells cease to proliferate. Dephosphorylated RB traps the transcription factors that are necessary for the generation of the cyclins required for cell proliferation, resulting in a cyclin-poor cell. When GSH is restored, E is decreased, RB can be phosphorylated and these cells proliferate (Yamauchi et al., 1997). This critical value of E which induces cessation of cell proliferation (CCP), is designated ECCP. Arrest in G1pm, the first part of the G1 phase of the cell cycle (the postmitotic interval of G1 that lasts from mitosis to the restriction point R), prevents the cell from proceeding to the second part of the G1 phase, G1ps, (the pre-S phase interval of G1 that lasts from R to S), as well as to S and to subsequent phases of the cell cycle. When this arrest has persisted for a few hours, then the duration required for apoptosis induction is achieved. Consequently, as the cancer cells that are in G1pm, are unable to enter G0 (Zetterberg et al., 1995), they will undergo apoptosis. In contrast, normal cells in G1pm can, and do, enter G0 and are able to stay there indefinitely. A model of the normal and cancer cell cycles is summarized in Scheme FIG. 1, which shows the cycle of a normal proliferating cell (black) and of a tumor cell (gray). Notice that the cell-cycle period for the tumor cell is shorter than that of the normal cell by the duration of G1 pm, which the tumor cell skips. The redox potential E is shown for each type of cell. The redox potential of the normal cell cycles between a high value during G1 pm and a low value during G2 and half of M. The transitions between these two levels are shown as dashed lines because of our lack of knowledge of the precise time profiles of these transitions. The redox potential of the tumor cell is constant throughout the cell cycle and is below the threshold Eccp. The redox potential of the normal proliferating cell cycles lies above and below the threshold.
Hutter et al. (1997) have studied the redox-state changes in density-dependent regulation of normal and malignant cell proliferation in the presence of modulators of GSH synthesis and have suggested a possible interrelationship between the redox potential and cell proliferation. Lee et al. (1998) showed that glucose deprivation-induced cytotoxicity is mediated by oxidative stress with formation of intracellular hydrogen peroxide in human breast carcinoma cells. Rossi et al. (1986) showed that the cytotoxicity of dimethyl- and trimethyl-benzoquinones to normal hepatocyte cells was due to a decrease in the [GSH] due to the formation of a quinone conjugate without oxidation to GSSG, while the addition of duroquinone, a tetramethylbenzoquinone, stimulated GSH oxidation and was only cytotoxic when catalase or glutathione reductase (GR) was inactivated. Smaaland et al., 1991, found a statistically significant correlation between the GSH content and the fraction of bone marrow cells in DNA synthesis.
There are many approaches for treating tumors. Some of these approaches are, to some extent, selective, such as the surgical removal of the tumor. In general, surgery is effective if the tumor has not spread and all the malignant cells have been removed. Other approaches are less selective and include radiation and chemotherapy, which usually affect normal cells as well. An agent is considered to provide a selective result if it mostly affects the cancer cells of the tumor, but does little, if any, harm to the adjacent normal cells of the tissue.
Many of the classical chemotherapeutic agents are usually more effective when the cancer cells in the tumor are rapidly proliferating. Some of the known cytotoxic agents such as vincristine, vinblastine, etoposide, methotrexate, 5-fluorouracyl, cytarabine, cisplatine, generally affect DNA during cell proliferation, primarily killing cancer cells rather than the relatively slowly proliferating normal cells. But this selectivity factor is not operative when treating slowly proliferating cancer cells. Other anti-cancer agents have been developed such tamoxifen, taxol, flavopiridol, angistatin, retinoic acid (all-trans and 9-cis), which do not affect the DNA during cell proliferation. Various mechanisms have been suggested for those two classes of agents, hereby designated as standard chemotherapeutic agents. There is, however, uncertainty in the conventional wisdom of the background art about the precise mechanisms involved. In general, anti-cancer agents, at their effective concentrations, are considered activators or triggers that trigger the formation of a sequence of various entities such as p21, which induce apoptosis (Li 1999; 2003). The concentrations of standard chemotherapeutic agents currently used for cancer treatment are limited usually to less than 5 μM (Ramachandran et al., 1999) in order to minimize injury to normal cells.
Reactive oxygen species (ROS), as generated by radiation, for example, are believed to cause mutations that produce cancer. There appears to be a consensus that antioxidants such as GSH, which can scavenge or otherwise neutralize the ROS, are required to prevent and treat cancer (Dai et al., 1999, Sen et al., 1999). If an antioxidant is defined as an agent that decreases E, by increasing the GSH2/GSSG ratio and, vice-versa, an oxidant as an agent that increases E, by decreasing the [GSH]2/[GSSG] ratio, some of the agents currently used as anticancer drugs or described in the literature as mentioned below, are clearly not acting as antioxidants.
In-vitro studies of treatment of tumor cell lines with several compounds have been carried out and have shown promising results, yet the basic mechanism of how these various compounds work remains obscure. In recent reports described hereinbelow, most experiments were performed with cell lines, that intrinsically involve relatively rapid cell proliferation, and the results with these various agents may not demonstrate their selectivity or their effectiveness in more slowly growing tumors.
Dai et al. (1999) introduced As2O3 into various cell lines. The resulting intracellular GSH content had a decisive effect on As2O3-induced apoptosis, the tendency to apoptosis increasing as the GSH content of the cell decreased. GSH forms an adduct with arsenic (As), viz., As(GS)3. These researchers experimentally varied the GSH content of the various cells with BSO (buthionine sulfoximine), which inhibits gamma-glutamylcysteine synthetase, GCS, a key enzyme in GSH biosynthesis. Tendency to apoptosis increased as GSH content decreased. By itself, BSO, which caused a decrease in [GSH] of 70% in the cell, did not induce significant apoptosis, but rendered the malignant cells more sensitive to As2O3. The authors did not report any measured value of [GSSG]. Normal cells showed the least apoptosis.
Nicole et al. (1998) showed that the introduction of BSO to neuroblastoma cells, decreasing their GSH content by 98%, and induced apoptosis. Here, too, they did not report any measured value of [GSSG]. They concluded that, with these cells, there was a cause-and-effect relationship between decreasing GSH and apoptosis induction.
Sen et al. (1999) introduced α-lipoic acid into both Jurkat T-cell leukemia cells and normal lymphocytes, and noticed that the leukemia cells underwent apoptosis, whereas the normal cells did not. They suggested that the induction of apoptosis by α-lipoic acid was because this acid is a sulfur-containing antioxidant that provides strong reducing power and leads to the reduction of protein thiols.
Lizard et al. (1998) reported that the introduction of 7-ketocholesterol to U937 cancer cells induced apoptosis. They found that apoptosis was enhanced by the addition of BSO and inhibited by the addition of NAC (N-acetyl-L-cysteine), a cysteine precursor which penetrates the cell and is converted by deacetylation to cysteine, which is a GSH precursor. The authors suggested that oxidative processes are involved in 7-ketocholesterol-induced cell death.
Rudra et al. (1999) reported that the introduction of acrolein induced cytotoxicity in various cancer cell lines, such as A-427 and A-172. They demonstrated that the sensitivity to growth inhibition increases as GSH decreases. They also reported that A-427 is highly sensitive to docosahexaenoic acid, and that acrolein potentiates the cytotoxic effect of this acid. These researchers reported that acrolein depletes thiols and is highly toxic to both normal human bronchial fibroblasts and human bronchial epithelial cells in the respiratory system.
Rossi et al, (1986), Thornton et al. (1995) and Cornwell et al (1998), introduced various quinones or quinone precursors to both normal cells, such as smooth muscle cells and hepatocytes, and to leukemic cells. Rossi et al. (1986) concluded that, when GSH decreased by 90-95% of the original amount in the hepatocytes, significant cytotoxicity was induced. They all concluded that the quinones formed a Michael Adduct with the GSH.
Ramachandran et al. (1999) introduced curcumin to both human mammary epithelial cells (MCF-10A) and breast carcinoma (MCF-7/TH) cell lines, and concluded that the induction of apoptosis is due to the effect of the curcumin on some of the genes associated with cell proliferation.
Zhou et al. (1998) introduced soy isoflavones to human prostate carcinoma cells and normal vascular endothelial cells. They suggested that these soy products inhibit experimental prostate tumor growth through a combination of direct effects on tumor cells and indirect effects on tumor neovasculature.
Paschka et al. (1998) induced apoptosis of prostate cancer cell lines by introducing green tea phenols including (−)-epigallocatechin-3-gallate.
With respect to tumors in general, especially slowly growing tumors, there is a dire need for agents that can selectively cause the cessation of cell proliferation (CCP), either as a result of cell arrest or apoptosis, similar to the effect of radiation on cells. Radiation is a p53 inducer, and the latter, in turn, induces p21, which can then combine with or otherwise inactivate the cyclins normally required for cell proliferation. As a result, the cyclin-poor cell undergoes cell cycle arrest or apoptosis (Gottlieb & Oren, 1996). In many cases, however, radiation is not completely selective, since it affects adjacent normal tissues; in addition, it causes unpleasant and serious side effects. Thus, more selective and effective treatments for cancer are required.
Throughout this specification, various scientific publications and patents or published patent applications are referenced. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of all these publications in their entireties is hereby incorporated by reference into this specification in order to more fully describe the state of the art to which this invention pertains. Citation or identification of any reference in this section or any other part of this application shall not be construed as an admission that such reference is available as prior art to the invention.